1,3-Diarylpyrazolyl-acylsulfonamides Target HadAB/BC Complex in Mycobacterium tuberculosis

Alternative mode-of-inhibition of clinically validated targets is an effective strategy for circumventing existing clinical drug resistance. Herein, we report 1,3-diarylpyrazolyl-acylsulfonamides as potent inhibitors of HadAB/BC, a 3-hydroxyl-ACP dehydratase complex required to iteratively elongate the meromycolate chain of mycolic acids in Mycobacterium tuberculosis (Mtb). Mutations in compound 1-resistant Mtb mutants mapped to HadC (Rv0637; K157R), while chemoproteomics confirmed the compound’s binding to HadA (Rv0635), HadB (Rv0636), and HadC. The compounds effectively inhibited the HadAB and HadBC enzyme activities and affected mycolic acid biosynthesis in Mtb, in a concentration-dependent manner. Unlike known 3-hydroxyl-ACP dehydratase complex inhibitors of clinical significance, isoxyl and thioacetazone, 1,3-diarylpyrazolyl-acylsulfonamides did not require activation by EthA and thus are not liable to EthA-mediated resistance. Further, the crystal structure of a key compound in a complex with Mtb HadAB revealed unique binding interactions within the active site of HadAB, providing a useful tool for further structure-based optimization of the series.


Methods: ESKAPE Antimicrobial Screening
The selectivity of compounds was assessed in a microdilution broth method against the selected Gram negative and one Gram positive strains, in accordance with the Clinical and Laboratory Standards Institute guidelines (CLSI, 2017 Overnight cultures of the different bacterial strains were grown on Müller-Hinton (MH) agar plates at 37˚C. Inoculum cultures were prepared in phosphate buffer saline (PBS) to a 0.5 McFarland (OD600 = 0.08) and diluted in MH broth to yield a bacterial concentration of approximately 1.5 x 10 6 cfu/mL. The compounds were tested at a concentration range between 0.25 -128 µM. Furthermore, three antibiotics (ciprofloxacin, gentamycin, moxifloxacin, and kanamycin) were used as controls. Untreated bacterial controls as well as media sterility controls were included in the assay. The prepared plates were incubated at 37°C for 16 hours, followed by a visual assessment. The minimum inhibitory concentration (MIC) values were determined as the lowest concentration where no bacterial growth could be observed.  . Chemoproteomic profiling using 3-bead-matrix Chemoproteomic profiling showed only a weak effect on HadC by 1,3-Diarylpyrazolylacylsulfonamide series compounds. Correlation of target binding and anti-mycobacterial activity for compounds from the 1,3-Diarylpyrazolyl-acylsulfonamide series. Experiments were performed as described in Fig 1. 5 compounds from the series with activity against Mtb and 3 that did not show activity (see table) were analyzed at 10 µM in two independent replicates on 3-bead-matrix. Active compounds showed inhibition of proteins HadA, HadB, BCG_0547c (HadD) and HadC from 3-bead-binding, while inactive compounds did not show any. HadA/B/D inhibition from bead-binding is in the range of 94-100%, while for HadC the inhibition is in a range of 52-64%, which indicates weaker potential IC50-values for HadC. r: Pearson correlation coefficient; p: p-value (calculated probability), Mtb pMIC defined as -log10(MIC99 in M).

Chemistry
Unless otherwise stated, the starting materials, reagents and solvents were purchased as high-grade commercial products. Analytical TLC was performed on Merck silica gel (60F254) precoated plates (0.25 mm). The compounds were visualized under UV light (254 nm) and/or stained with the relevant reagent. Flash column chromatography was performed on silica gel with pore size 60 Å, 230−400 mesh particle size, and 40−63 μm particle size, with the indicated solvents. The yields refer to the purified products, and they were not optimized. All the solid compounds were obtained as amorphous solids, and melting points were not measured. 1 H NMR spectra were recorded on a Bruker Avance III NMR spectrometer and in a Bruker DPX Avance 400 MHz instrument equipped with a QNP probe and are reported in ppm using tetramethylsilane as internal standard. Mass spectra data measurements were performed on a VG-Analytical Autospec Q mass spectrometer. Analytical purity was ≥95% unless stated otherwise. The purities of the final compounds were determined by using a Waters ZQ2000 coupled with LC Waters 2795 and Waters 2996 PDA detector.

High-resolution mass method:
HPLC-HRMS was obtained using an AB Sciex® X500R QTOF coupled to an AB Sciex® Exion LC system. Spectral data were obtained using information dependent acquisition (IDA) at a mass range of 50-1200 Da. All methods, batches and data were processed using OS Sciex® v1.2. The declustering potential was 80 V, the curtain gas (N2) was at 25 pounds per square inch (psi), the ion spray voltage was 5500 V, and the source temperature was 450°C. Ion source gases 1 and 2 were at 45 and 55 psi, respectively. The collision energy was 10 eV for the MS scans and 20-50 eV for MS/MS scans. The IDA intensity threshold was 50 cycles per second. The aqueous mobile phase used was 1 mM ammonium formate in water, and the organic mobile phase was methanol with 0.5% formic acid. The flow rate was 700 µL/minute, and the method ran from 2% to 98% organic for 25 minutes, was held at 98% organic for a further 2 minutes before returning to 2% organic over 3 minutes to equilibrate for the next run. A Kinetex® C18 LC column (5 µm, 100 Å, 150 mm × 4.6 mm) with a column protector was used. A blank injection was run between each sample. The spectra were processed and displayed using MZMine2.5. 1 S11 Scheme S1. Synthetic pathway for compounds 3 and 4.

1-(4-chlorophenyl)pentane-1,3-dione (25):
A solution of propionic acid (1.01 mL, 13.50 mmol) and CDI (3.38 g, 20.25 mmol) in anhydrous DMF (10 mL) was allowed to stir at room temperature under N2 atmosphere for 3 hours. The mixture was added portion wise to a vigorous stirring suspension of 1-(4-chlorophenyl)ethan-1-one (24, 1.751 mL, 13.50 mmol) and sodium hydride 60% suspension in mineral oil (1.620 g, 40.5 mmol) in DMF (10 mL). The resulting reaction mixture was allowed to stir at 80 °C under N2 atmosphere for 15 hours. The reaction mixture was allowed to cool at room temperature and poured into ice-cold and neutralised with 2 N HCl. The obtained aqueous layer was extracted with EtOAc (4 x 25 mL). The combined organic layers were washed with water, followed by brine, dried over MgSO4, filtered and solvent evaporated in vacuo to yield a brown residue. The obtained residue was adsorbed into silica gel and purified using an ISCO Teledyne CombiFlash system (40 g SiO2 cartrige) eluting a gradient of EtOAc in hexane (hexane 100% to hexane/EtOAc 95:5). Pure fractions were combined to yield 25 (0.492 g, 2.195 mmol, 16% yield) a yellow liquid. Intermediate used in the next step without further purification. MS-ES + [M+H] + = 211.0

1,3-bis(4-chlorophenyl)-5-ethyl-1H-pyrazole-4-carbaldehyde (28):
To a stirring solution of 27 (236 mg, 0.744 mmol) in DMF (15 mL) was added POCl3 (2.08 mL, 22.32 mmol) slowly at ambient temperature and the resulting reaction mixture was heated at 80 °C for 19 hours. The reaction mixture was cooled and poured onto ice-water and basified to pH=8 with NaOH. The obtained aqueous layer was extracted with EtOAc (4 x 15 mL). The combined organic layer was washed with H2O (10 mL) followed by brine (10 mL), then dried over MgSO4, filtered and solvent evaporated in vacuo to yield a crude product. The crude product was adsorbed into silica gel and purified using an ISCO Teledyne CombiFlash system (25 g SiO2 cartridge) eluting a gradient of EtOAc in hexane (hexane 100% to hexane/EtOAc 85:15) to afford 28 (109 mg, 0.316 mmol, 42 % yield) as a yellow solid. Intermediate was used in the next step without further purification. 1

(1,3-bis(4-chlorophenyl)-5-ethyl-1H-pyrazol-4-yl)methanol (29):
In an ice-cold water bath, a solution of 28 (109 mg, 0.316 mmol) in methanol (15 ml) was added sodium borohydride (23.89 mg, 0.631 mmol) portion wise and the resulting reaction mixture was allowed to stir at room temperature for 1 hour. LC-MS indicated complete consumption of the starting material. The reaction was quenched by careful addition of H2O (10 mL) and solvent was removed in vacuo to yield a residue. The residue obtained was diluted with water and extracted with EtOAc (4 x 15 mL), the combined organic layers were washed with brine (15 mL), dried over MgSO4, filtered and solvent evaporated in vacuo to afford 29 (76 mg, 0.193 mmol, 61 % yield) as a yellow solid. Intermediate was used in the next step without further purification. MS-ES + [M+H] + = 347.0.
(1,3-bis(4-chlorophenyl)-5-ethyl-1H-pyrazol-4-yl)methyl (methylsulfonyl)carbamate (15): A pressure tube containing a solution of N-ethyl-Nisopropylpropan-2-amine (0.310 mL, 1.774 mmol), methanesulfonamide (42.2 mg, 0.443 mmol) and di(1H-imidazol-1-yl)methanone (71.9 mg, 0.443 mmol) in DCM (5 mL) was allowed to stir at room temperature for 16 hours. To this solution was added 28 (77 mg, 0.222 mmol) in DCM (5 mL) and the resulting reaction mixture was allowed to stir at 70 °C for 24 hours. At the end, the reaction mixture was cooled to room temperature, diluted with water and extracted with DCM (4 X 20 mL). The combined organic layers were washed with brine (20 mL), dried over MgSO4, filtered and solvent evaporated in vacuo to afford a residue. The obtained residue was adsorbed into silica gel and purified using an ISCO Teledyne CombiFlash system (12 g SiO2 cartridge) eluting a gradient of EtOAc in hexane (hexane 100% to hexane/EtOAc 30:70) to afford 15 (30 mg, 0.062 mmol, 28 % yield) as a white solid. 1   Electron density is displayed as mesh, with a 5σ cutoff. The HadA subunit ribbon is colored yellow while the B subunit ribbon is colored green with the ligand carbons colored cyan. Figure S4. 3D illustrations of the binding modes of butein (A) and fisetin (B) the HadAB complex -PDB ID: 4RLW and 4RLT respectively. Butein (A) interacts predominantly with the S1 subsite forming π-interactions with A-Y65 and hydrogen bonds to A-Q89 and A-Q86. Hydrogen bonds to A-N126 and A-T138 are present in the S4 subsite. Hydrogen bonds with two water molecules interacting with the catalytic dyad of B-D-36 and B-H41 are observed but no direct interactions with the catalytic hooks of the S2 main chain amides of A-Q86 and B-G59. These interactions are crucial for positioning the substrate for the dehydratase reaction catalysed by the B-D36/B-H41 dyad. (B) Fisetin, a coumarin also interacts predominantly with the S2 and S4 subsites. The coumarin group occupies the S4 cavity, forming hydrogen bonds with 3 water molecules, B-N125 and B-Q68. The dihydroxyphenyl group interacts with the S1 subsite via a π-interactions with A-Y65 and a hydrogen bond with lone water, not engaging with the water network around the catalytic dyad or the catalytic hooks of the A-Q86 and B-G59 main chain amides. Figure S5. The unliganded structure or the MtbHadBC (HadB -light green, HadCorange) superimposed onto the crystal structure of MtbHadAB (HadAyellow, HadB lime green) in complex with 9 (turquoise). The two HadB subunits are shown to be identical while the HadC and HadB subunits are identical in the S2 subsite that interacts with the acyl sulfonamide moiety. The big difference occurs in the S3 subsite with the A-Y65 for C-K65 change. The apo form of the HadBC complex is also unsuitable for ligand docking due to the flexible C-K65 interfering with the ligand binding and a poorly defined region of the S2 subsite around C-I84 (circled in red).